A major objective of bioanalytical chemistry is to detect small amounts of DNA or RNA in samples. These samples are, of course, routinely found in research laboratories. However, they are also found in the clinic, where they can be obtained from human patients, or in veterinary medicine, where they come from animals, or in public places (such as a post office), where they might indicate the presence of an infectious disease, or on the battlefield, where they might indicate that an adversary is using a biological warfare agent. As one moves farther from the research laboratory, the desired specifications become more demanding:    1. Sensitivity. The ultimate goal is to detect single molecules of the analyte nucleic acid, preferably without needing to amplify it (as is presently done using the polymerase chain reaction).    2. Selectivity. The ultimate goal is to detect the analyte in complex biological material, including an environment that may contain billions of other oligonucleotide sequences.    3. Cost. A cheaper analytical tool is nearly always preferable to a more expensive tool.    4. Convenience. Ideally, detection should be done in a single step.    5. Readout. The readout must be simple, recognizing that simplicity is defined in many cases by the goal. On the battlefield, for example, a visual readout is desirable. In a trap seeking the Hanta virus in rodents, for example, an electrical readout might be desired.    6. Multiplexing. It is often desirable to be able to detect multiple oligonucleotide analytes at the same time.    7. Time. A read-out in seconds or minutes is preferable to read-outs that require more time to obtain.    8. Sample preparation. None is generally desirable.    9. Reproducibility is preferably be high; false positives are preferably low.    10. Dynamic range. The ability to quantitate from a single molecule to hundreds of thousands has value in specific circumstances.
The binding of DNA, RNA and DNA analogs (collectively called “probes”) to targeted natural DNA and RNA (called the “analyte” molecules, herein referred to as “N”) offers a step in a process that might be used to detect natural analyte molecules and, thereby, might support an assay that infers the presence of the living system that carries that analyte. Probes have been used for 40 years for this purpose, with their use accelerating as synthetic DNA became readily available in the early 1980's.
A variety of architectures are available to exploit the binding of analyte molecule to a probe molecule to generate a detectable readout as a consequence of that binding. These can be classified in various ways. One classification counts the number of probe molecules that are used in the assay. Thus, many architectures for detecting DNA sequences use a single probe molecule. Another class of architectures, however, uses the binding between an analyte and two probe molecules to generate the readout. Here, the most common feature allows the analyte molecule to bring the two probe molecules together in space. Once so arranged, the proximity between the two probes is then used as the basis for the generation of the read-out.
One well known example of this architecture is the “binary beacon”. Here, one of the two probes brought into proximity carries a fluorescence energy donor, while the second carries a fluorescence energy acceptor. Only when the two are held in proximity by hybridization to the analyte is fluorescent resonant energy transferred efficiently between the two. This process was demonstrated some time ago by Cardullo et al. [Car88] in vitro (see also review by [Tsi93]). Tsuji et al. used the FRET strategy to observe human c-fos mRNA inside a living transfected Cos7 cell [Tsu01]. With the efficiency of fluorescent resonance energy dropping with the sixth power of the distance between the energy donor and the energy acceptor, close proximity effected by having both molecules bind to the probe is an important contributor to the efficiency.
This work also illustrated many reasons why this analytical task is difficult. First, the secondary structure of an mRNA (if this is the analyte) itself is problematic. These workers identified by experiment a 40 mer sequence that hybridized with high efficiency to probes 15-20 nucleotides in length. To prevent the tagged DNA probes from accumulating in the nucleus, they bound them to streptavidin, which allowed them to be stable in the cytoplasm for over 30 minutes. This is, of course, a problem general to any method that hopes to detect an analyte by exploiting a probe.
Other issues relate to the sensitivity of this approach. Even without resonant energy transfer, a small amount of fluorescence is generally created by the second fluor even if the excitation wavelength is not optimally suited for direct excitation. This creates background noise. Thus, Tsuji et al. microinjected approximately 105 molecules into the cells that they studied, and were able to detect a specific mRNA only if it was present at ca. 10,000 copies of per cell [Tsu01]. They noted that both the fluorescence of the unhybridized fluorescent molecules introduced into the cell, as well as background autofluorescence of components naturally in the cell, limited the signal-to-noise ratio that might be achieved by this approach. They noted that the latter might be reduced by examining fluorescence species that absorb and emit at longer wavelengths. To seek the FRET signal above the noise arising from unhybridized fluroescent probes, Tsuji et al. also explored time-resolved fluorescence spectroscopy [Tsu01].
Fluorescence readout is not the only way to generate a read-out as a consequence of the binding of two probes in adjacent positions on an analyte molecule. For example, such binding might lead to the formation of a covalent bond between the two probe molecules. Examples of this process have been available for over 20 years [Kie86]. More recently, Xu and Kool [Xu97] replaced the 5′-hydroxyl group on a DNA fragment by an iodo group on the 5′-end of one probe, and placed a phosphorothioate on the 3′-end of the second probe. Binding the two to an analyte molecule N on adjacent segments of the oligonucleotides sequence brought the reactive electrophilic center (the carbon attached to the iodine) into proximity to the reactive nucleophilic center, and facilitated their reaction to make a covalent phosphothioate diester bond. Here, the reaction is faster because the two species having complementary reactivity are held together by hybridization to the analyte.
While in widespread use, such strategies suffer from the intrinsic background reaction between nucleophile and electrophile as they come into contact in the absence of the analyte molecule. This renders most of the architectures laboratory curiosities.
As recently as 2002, various groups have attempted to carve out intellectual property from this basic idea. For example, Liu, Gartner and Calderone (United States Patent Application 20040180412) have claimed a method of inducing reaction between first and second reactive units attached to oligonucleotides probes by using a nucleic acid-templated chemical reaction. Their method comprised the steps of: (a) providing (i) a template comprising a first reactive unit associated with a first oligonucleotide comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon capable of annealing to said codon, wherein said codon or said anti-codon comprise first and second spaced apart regions; (b) annealing said oligonucleotides together thereby to bring said first reactive unit and said second reaction unit into reactive proximity, wherein said codon or said anti-codon having said first and second spaced apart regions produce a loop of oligonucleotides not annealed to the corresponding anti-codon or codon; and (c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product. This was proposed to be a tool for generating libraries of organic molecules for the purpose of chemical screening.
A consideration of thermodynamics provides a theoretical perspective on such approaches, insight into why they will not have high utility as they are presently implemented, and ideas for alternative implementations that are more likely to be useful. First, if the untemplated joining of two molecules is to be avoided, energy that drives (either from a kinetic or a thermodynamic perspective) the ligation reaction cannot be intrinsically present in the reactive species at the ends of the probes. Rather, this energy must be introduced from time to time to create an activated probe ready to form a covalent bond with the capture probe, and then be dissipated. The rate of energy dissipation must be slow enough to allow the formation of the desired covalent link between the two probe molecules if they are preorganized, held together by binding to the analyte. If they are not pre-organized, however, however, the energy must be dissipated before the activated probe molecule finds its reaction partner by diffusion in solution. This can be had by having the activated moiety react harmlessly with bulk assay components (for example, the water in the assay).
Further, the architecture is most effectively implemented if the dissipation restores the activated probe to a state identical to that before activation. This conserves the probe, allowing it to have another opportunity to be activated again in the future, when it is possibly bound near its capture probe to an analyte molecule.
This activation can, of course, come from adding a chemical reagent. Several of these are contemplated in the invention described below. The preferred implementation of this strategy, however, is use of a photon to activate an activatable moiety on one of the probes.